Tumorigenesis resembles abnormal embryogenesis. Like developing organs, tumors comprise a spectrum of phenotypically heterogeneous cells and may harbor stem cell-like cells commonly referred to as cancer stem cells (CSCs). Although the CSC concept is still debated and the true molecular nature and clinical significance of CSCs remain to be fully elucidated, it is undeniable that malignant tumors are immortal at the population level. One of the key unanswered biological questions of therapeutic implication is how cancer cells in general and CSCs in particular are regulated at the molecular level with respect to their self-perpetuating (immortal) tumorigenic potential, i.e., self-renewal properties. Remarkably, malignant cells share a similar gene expression profile with embryonic SC (ESCs), which express several core regulators of self-renewal and pluripotency including Nanog (also called Nanog1), a homeodomain transcription factor located on chromosome 12. We have observed that human PCa and other cancer cells do NOT express Nanog1 but rather express a retrotransposed Nanog1 homolog, called NanogP8, located on chromosome 15. Knocking down NanogP8 significantly inhibits the CSC properties in vitro and tumor regeneration of PCa and breast and colon cancer cells. In contrast, NanogP8 overexpression is sufficient to confer CSC properties, enhances tumor regeneration, and, significantly, promotes development of castration-resistant PCa or CRPC. Our recent whole-genome ChIP-Seq experiment has uncovered distinct NanogP8 DNA-binding profiles. Furthermore, newly generated NanogP8 transgenic animals develop hyperplastic lesions in the target organs. These observations, taken together, lead to our overarching hypotheses that 1) NanogP8 possesses protumorigenic activity; 2) NanogP8 promotes tumor development by regulating CSC self-renewal via unique signaling pathways; and 3) NanogP8 plays a causal role in the development of CRPC. The current project tests these hypotheses, mainly, in PCa with the following three Specific Aims. 1) To test the hypothesis that NanogP8 is causally involved in the development of CRPC; 2) To elucidate the cellular and molecular mechanisms of action of NanogP8; and 3) To explore the protumorigenic roles of NanogP8 using novel transgenic animal models. Impact: Accomplishment of the goals proposed herein should greatly advance our understanding of how NanogP8 regulates the immortality of CSCs and contribute to tumor development and CRPC. It will also facilitate development of mechanism-based therapeutics that specifically targets PCSCs. PUBLIC HEALTH RELEVANCE: It is generally unclear how cancer cells acquire their immortal nature. We have recently demonstrated that cancer cells appear to usurp a molecule critical for the self-renewal of embryonic stem cells (ESCs) to help achieve their immortality. Specifically, ESCs express a protein called Nanog1 localized on chromosome 12 for their self-renewal. By contrast, cancer cells preferentially express a retrotransposed homolog of Nanog1 called NanogP8, which is localized on chromosome 15 and is required for the tumorigenic potential of cancer cells. In this project, we aim to further elucidate the molecular functions and mechanisms of NanogP8 in cancer (mainly prostate cancer) cells.